EXPERIMENTAL AND THERAPEUTIC MEDICINE 12: 589-596, 2016
Expression of MCRS1 and MCRS2 and their correlation with serum carcinoembryonic antigen in colorectal cancer CHENGUANG LI1,2, MINGXIAO CHEN1, PINGWEI ZHAO1, DESALEGN ADMASSU AYANA3, LEI WANG1 and YANFANG JIANG2 1
Department of Colorectal and Anal Surgery, The First Hospital, Jilin University, Changchun, Jilin 130032; 2 Key Laboratory of Zoonosis Research, Ministry of Education, The First Hospital, Jilin University, Changchun, Jilin 130032, P.R. China; 3Department of Medical Laboratory Sciences, Haramaya University, Dire Dawa 3000, Ethiopia Received June 30, 2015; Accepted March 3, 2016 DOI: 10.3892/etm.2016.3424
Abstract. Cancer‑associated genes serve a crucial role in carcinogenesis. The present study aimed to investigate the mRNA expression levels of microspherule protein 1 (MCRS1) and MCRS2 in colorectal cancer (CRC) and their association with clinical variables. The mRNA expression levels of MCRS1 and MCRS2 were assessed by semi‑quantitative reverse transcription polymerase chain reaction in the tumor and corresponding non‑tumor tissues of 54 newly‑diagnosed CRC patients, as well as in the normal colonic mucosa tissue of 19 age/gender‑matched healthy controls. Immunofluorescence was also employed to identify the expression of MCRS1 in CRC tissues, while the concentration of serum carcinoembryonic antigen (CEA) was determined by an enzyme‑linked immunoassay. The results identified a negative correlation between MCRS1 and MCRS2 expression levels (r=‑0.3018, P=0.0266). MCRS1 mRNA expression was significantly increased and MCRS2 mRNA expression was decreased in CRC tissues compared with the levels in the corresponding normal tissues (both P1.2 million individuals globally each year (1). CRC is the third most common cancer and the leading cause of cancer‑associated mortality in the United States (1,2) and the incidence of CRC is increasing in China (3‑5). The morphological changes involved in the progression of CRC from a benign adenoma to a malignant carcinoma have a complex biological and molecular underlying process (6). Cancer‑associated genes serve an important role in carcinogenesis, including the processes of cell proliferation, transformation, angiogenesis, invasion and metastasis. The increased activity of oncogenes or decreased activity of tumor‑suppressor genes have been found to participate in the development of tumors (7). A number of CRC cases exhibit mutations in the KRAS oncogene (8‑11), as well as in p53 (12), APC and SMAD4/DPPC4 (13‑15) tumor‑suppressor genes. Furthermore, a large number of mutated genes have been identified in CRC, including genes that encode proteins with vital roles in CRC carcinogenesis. However, the molecular mechanism of CRC carcinogenesis has yet to be elucidated. The major cause of CRC mortality and morbidity is metastasis, with >30% of patients with CRC eventually developing metastatic disease (16‑18). Thus, there is an urgent requirement for specific and sensitive biomarkers to aid the early diagnosis and prognosis of CRC. Microspherule protein 1 (MCRS1), also known as MSP58, is involved in various pivotal cellular processes, such as DNA repair, regulation of the cell cycle, development of malignancy, transcription and mitosis (19‑21). MCRS1 is a
590
LI et al: MCRS1 AND MCRS2 EXPRESSION IN COLORECTAL CANCER
462‑amino acid protein which was originally identified as the interaction partner of the p120 nucleolar protein (22), and its overexpression results in the enlargement of nucleoli. Studies have indicated that the interaction of MCRS1 with the transcription factors Daxx (23), STRA13 (24), REP (25), and REP (24) confirmed the function of regulation in transcriptional activity. Increasing evidence has demonstrated that MCRS1 interacts with various proteins that serve critical roles in tumor proliferation; for instance, it is reported that p78, an isoform of MCRS1, has been identified as a centrosomal protein that is required for maintaining centrosome homeostasis (26). Notably, MCRS1 has been identified to be highly expressed in CRC (27), hepatocellular carcinoma (28), glioma (29‑31) and non‑small‑cell lung cancer (32), and to be correlated with poor prognosis. The transformation activity of MCRS1 is inhibited by physical interaction with the PTEN tumor suppressor, with the majority of evidence suggesting that MCRS1 behaves as an oncogene (31,33). By contrast, MCRS2 has been identified as an interacting partner and a potent inhibitor of telomerase, and long‑term overexpression of MCRS2 in cancer cell lines leads to telomere shortening (34‑36). Drosophila MCRS2 is co‑purified with RNA polymerase II complexes and is required for normal levels of cycling gene expression (37). A previous study revealed that MCRS1 and MCRS2 are involved in cell cycle regulation and carcinogenesis (20,32). Carcinoembryonic antigen (CEA) was first identified in 1965 (38) and has now become the most widely used antigen for diagnosing and for monitoring the prognostic significance of CRC (39). However, a lack of sensitivity renders its use limited in clinical diagnosis (40). Thus, it is important to identify novel biomarkers and develop novel treatment strategies for CRC. Based upon the crucial role of MCRS1 and MCRS2 in tumorigenesis, semi‑quantitative reverse transcription polymerase chain reaction (RT‑PCR) was used in the present study to detect the mRNA expression levels of MCRS1 and MCRS2 in 54 fresh tissue samples from patients with CRC, along with their corresponding normal tissues. MCRS1 expression was also detected by immunofluorescence. In addition, the association of MCRS1 and MCRS2 expression with the clinicopathological features of CRC was also assessed. MCRS1 and MCRS2 expression and the clinical variables were analyzed in order to evaluate the clinical significance of differences in the gene expression levels. Materials and methods Patients and tissues. A total of 54 newly‑diagnosed CRC patients at the inpatient service of the First Hospital of Jilin University (Changchun, China) were recruited into the study between April 2011 and August 2013. In addition, 19 healthy individuals were recruited as controls. Written informed consent was obtained from individual participants. The experimental protocol was established according to the Declaration of Helsinki (sixth revision, 2008) and approved by the Medical Ethics Committee of the First Hospital of Jilin University. The baseline demographic and clinical data of individual participants were collected from hospital records and reviewed by experienced surgeons. The demographic and clinical characteristics of participants were obtained.
Patients were initially screened with fecal occult blood test (Benzidine test) to detect potential bleeding in the digestive tract. A piece of faeces was emulsified and 1 ml saturated benzidine solution (Sangon Biotech, Co., Ltd., Shanghai, China) was added. Thereafter, hydrogen peroxide was slowly added followed by vigorous shaking of the solution. A color change from green to blue was regarded as a positive result, whereas, a change to deep purple without first becoming green or blue was considered to be a negative result. Furthermore, patients were diagnosed following the histological examination of biopsy [hematoxylin and eosin (HE) staining (Beijing Zhongshan Golden Bridge Biotechnology, Co., Ltd., Beijing, China) or immunohistochemical staining (with Bcl‑2, Her‑2, EGFR, p53, Ki‑67; Fuzhou Maixin Biotech. Co., Ltd., Fuzhou, China) or both]. Tumor tissues were obtained during colonoscopy with the video endoscopy system Olympus Evis Lucera Elite (Olympus, Tokyo, Japan), which was followed by a computed tomography scan (Brilliance CT 64‑slice, Philips, Holland). The tumor classification, histological grades and lymph node metastasis status of individual tumor samples were evaluated by pathologists in a blinded manner, and staged according to the tumor‑node‑metastasis (TNM) classification system of the International Union against Cancer (edition 7). (41). Patients with stage I/II of CRC were classified as early stage patients while those with stage III/IV of CRC were grouped as advanced stage patients. A total of 54 freshly resected surgical tumour tissues and the corresponding non‑tumor tissues that were obtained from a location ≥5 cm away from the centre of CRC were assessed by HE staining. Moreover, 19 normal mucosa samples were obtained from patients with hemorrhoids when they underwent a procedure for treatment of hemorrhoids at the same department. These healthy control patients had no other gastrointestinal disease and their tissue samples served as the healthy control tissues (Table I). Individual patients were excluded if they had a history of previous tumor and received radiotherapy or chemotherapy, poor physical condition, undergone treatment with immunosuppressants during the previous three months or were >80‑years‑old. Normal colonic mucosa, fresh tumor tissues and the corresponding non‑tumor control tissues were obtained from patients during surgery and were used for RNA extraction. Semi‑quantitative RT‑PCR. Total RNA was extracted from the normal colonic mucosa, fresh surgical tumor and non‑tumor tissue samples using the Eastep Super Total RNA Extraction kit (LS1000, Promega Corp., Shanghai, China), according to the manufacturer's protocol. RNA quantity and quality were assessed using a Synergy HTX Multi‑Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA) and the absorbance ratio of 260/280 nm should have been in the range between 1.8 to 2.0. The integrity of RNA was confirmed using a 2.0% agarose gel electrophoresis and visualized with a Tanon‑2500R Gel Imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China). Electrophoresis of RNA on the agarose gel demonstrated the appearance of a 5S band, and clear 28S and 18S bands that suggested the integrity of RNA. The integrity of RNA was confirmed using agarose gel electrophoresis. First strand cDNA was obtained using the SuperScript II Reverse Transcriptase
EXPERIMENTAL AND THERAPEUTIC MEDICINE 12: 589-596, 2016
591
Table I. Demographic and clinical characteristics of subjects. Characteristic
Healthy controls (n=19)
Non‑tumor controls (n=54)
Early CRC (n=23)
Advanced CRC (n=31)
Mean age (range), years 55 (43‑69) 58 (44‑71) 57 (44‑71) 60 (48‑70) Gender (male/female) 11/8 32/22 13/10 19/12 Tumor location (colon/rectum) N/A 21/33 8/15 13/18 TNM stage (I/II or III/IV) N/A N/A 7/16 (I/II) 21/10 (III/IV) Differentiation (good/moderate/poor) N/A N/A 7/9/7 5/11/15 Serum CEA, ng/ml 0.36 (0.15‑4.58) 7.24 (0.17‑38.34)a 4.37 (0.17‑26.74) 21.16 (2.7‑38.34)a Data are presented as the median and range unless otherwise specified. aP
Expression of MCRS1 and MCRS2 and their correlation with serum carcinoembryonic antigen in colorectal cancer CHENGUANG LI1,2, MINGXIAO CHEN1, PINGWEI ZHAO1, DESALEGN ADMASSU AYANA3, LEI WANG1 and YANFANG JIANG2 1
Department of Colorectal and Anal Surgery, The First Hospital, Jilin University, Changchun, Jilin 130032; 2 Key Laboratory of Zoonosis Research, Ministry of Education, The First Hospital, Jilin University, Changchun, Jilin 130032, P.R. China; 3Department of Medical Laboratory Sciences, Haramaya University, Dire Dawa 3000, Ethiopia Received June 30, 2015; Accepted March 3, 2016 DOI: 10.3892/etm.2016.3424
Abstract. Cancer‑associated genes serve a crucial role in carcinogenesis. The present study aimed to investigate the mRNA expression levels of microspherule protein 1 (MCRS1) and MCRS2 in colorectal cancer (CRC) and their association with clinical variables. The mRNA expression levels of MCRS1 and MCRS2 were assessed by semi‑quantitative reverse transcription polymerase chain reaction in the tumor and corresponding non‑tumor tissues of 54 newly‑diagnosed CRC patients, as well as in the normal colonic mucosa tissue of 19 age/gender‑matched healthy controls. Immunofluorescence was also employed to identify the expression of MCRS1 in CRC tissues, while the concentration of serum carcinoembryonic antigen (CEA) was determined by an enzyme‑linked immunoassay. The results identified a negative correlation between MCRS1 and MCRS2 expression levels (r=‑0.3018, P=0.0266). MCRS1 mRNA expression was significantly increased and MCRS2 mRNA expression was decreased in CRC tissues compared with the levels in the corresponding normal tissues (both P1.2 million individuals globally each year (1). CRC is the third most common cancer and the leading cause of cancer‑associated mortality in the United States (1,2) and the incidence of CRC is increasing in China (3‑5). The morphological changes involved in the progression of CRC from a benign adenoma to a malignant carcinoma have a complex biological and molecular underlying process (6). Cancer‑associated genes serve an important role in carcinogenesis, including the processes of cell proliferation, transformation, angiogenesis, invasion and metastasis. The increased activity of oncogenes or decreased activity of tumor‑suppressor genes have been found to participate in the development of tumors (7). A number of CRC cases exhibit mutations in the KRAS oncogene (8‑11), as well as in p53 (12), APC and SMAD4/DPPC4 (13‑15) tumor‑suppressor genes. Furthermore, a large number of mutated genes have been identified in CRC, including genes that encode proteins with vital roles in CRC carcinogenesis. However, the molecular mechanism of CRC carcinogenesis has yet to be elucidated. The major cause of CRC mortality and morbidity is metastasis, with >30% of patients with CRC eventually developing metastatic disease (16‑18). Thus, there is an urgent requirement for specific and sensitive biomarkers to aid the early diagnosis and prognosis of CRC. Microspherule protein 1 (MCRS1), also known as MSP58, is involved in various pivotal cellular processes, such as DNA repair, regulation of the cell cycle, development of malignancy, transcription and mitosis (19‑21). MCRS1 is a
590
LI et al: MCRS1 AND MCRS2 EXPRESSION IN COLORECTAL CANCER
462‑amino acid protein which was originally identified as the interaction partner of the p120 nucleolar protein (22), and its overexpression results in the enlargement of nucleoli. Studies have indicated that the interaction of MCRS1 with the transcription factors Daxx (23), STRA13 (24), REP (25), and REP (24) confirmed the function of regulation in transcriptional activity. Increasing evidence has demonstrated that MCRS1 interacts with various proteins that serve critical roles in tumor proliferation; for instance, it is reported that p78, an isoform of MCRS1, has been identified as a centrosomal protein that is required for maintaining centrosome homeostasis (26). Notably, MCRS1 has been identified to be highly expressed in CRC (27), hepatocellular carcinoma (28), glioma (29‑31) and non‑small‑cell lung cancer (32), and to be correlated with poor prognosis. The transformation activity of MCRS1 is inhibited by physical interaction with the PTEN tumor suppressor, with the majority of evidence suggesting that MCRS1 behaves as an oncogene (31,33). By contrast, MCRS2 has been identified as an interacting partner and a potent inhibitor of telomerase, and long‑term overexpression of MCRS2 in cancer cell lines leads to telomere shortening (34‑36). Drosophila MCRS2 is co‑purified with RNA polymerase II complexes and is required for normal levels of cycling gene expression (37). A previous study revealed that MCRS1 and MCRS2 are involved in cell cycle regulation and carcinogenesis (20,32). Carcinoembryonic antigen (CEA) was first identified in 1965 (38) and has now become the most widely used antigen for diagnosing and for monitoring the prognostic significance of CRC (39). However, a lack of sensitivity renders its use limited in clinical diagnosis (40). Thus, it is important to identify novel biomarkers and develop novel treatment strategies for CRC. Based upon the crucial role of MCRS1 and MCRS2 in tumorigenesis, semi‑quantitative reverse transcription polymerase chain reaction (RT‑PCR) was used in the present study to detect the mRNA expression levels of MCRS1 and MCRS2 in 54 fresh tissue samples from patients with CRC, along with their corresponding normal tissues. MCRS1 expression was also detected by immunofluorescence. In addition, the association of MCRS1 and MCRS2 expression with the clinicopathological features of CRC was also assessed. MCRS1 and MCRS2 expression and the clinical variables were analyzed in order to evaluate the clinical significance of differences in the gene expression levels. Materials and methods Patients and tissues. A total of 54 newly‑diagnosed CRC patients at the inpatient service of the First Hospital of Jilin University (Changchun, China) were recruited into the study between April 2011 and August 2013. In addition, 19 healthy individuals were recruited as controls. Written informed consent was obtained from individual participants. The experimental protocol was established according to the Declaration of Helsinki (sixth revision, 2008) and approved by the Medical Ethics Committee of the First Hospital of Jilin University. The baseline demographic and clinical data of individual participants were collected from hospital records and reviewed by experienced surgeons. The demographic and clinical characteristics of participants were obtained.
Patients were initially screened with fecal occult blood test (Benzidine test) to detect potential bleeding in the digestive tract. A piece of faeces was emulsified and 1 ml saturated benzidine solution (Sangon Biotech, Co., Ltd., Shanghai, China) was added. Thereafter, hydrogen peroxide was slowly added followed by vigorous shaking of the solution. A color change from green to blue was regarded as a positive result, whereas, a change to deep purple without first becoming green or blue was considered to be a negative result. Furthermore, patients were diagnosed following the histological examination of biopsy [hematoxylin and eosin (HE) staining (Beijing Zhongshan Golden Bridge Biotechnology, Co., Ltd., Beijing, China) or immunohistochemical staining (with Bcl‑2, Her‑2, EGFR, p53, Ki‑67; Fuzhou Maixin Biotech. Co., Ltd., Fuzhou, China) or both]. Tumor tissues were obtained during colonoscopy with the video endoscopy system Olympus Evis Lucera Elite (Olympus, Tokyo, Japan), which was followed by a computed tomography scan (Brilliance CT 64‑slice, Philips, Holland). The tumor classification, histological grades and lymph node metastasis status of individual tumor samples were evaluated by pathologists in a blinded manner, and staged according to the tumor‑node‑metastasis (TNM) classification system of the International Union against Cancer (edition 7). (41). Patients with stage I/II of CRC were classified as early stage patients while those with stage III/IV of CRC were grouped as advanced stage patients. A total of 54 freshly resected surgical tumour tissues and the corresponding non‑tumor tissues that were obtained from a location ≥5 cm away from the centre of CRC were assessed by HE staining. Moreover, 19 normal mucosa samples were obtained from patients with hemorrhoids when they underwent a procedure for treatment of hemorrhoids at the same department. These healthy control patients had no other gastrointestinal disease and their tissue samples served as the healthy control tissues (Table I). Individual patients were excluded if they had a history of previous tumor and received radiotherapy or chemotherapy, poor physical condition, undergone treatment with immunosuppressants during the previous three months or were >80‑years‑old. Normal colonic mucosa, fresh tumor tissues and the corresponding non‑tumor control tissues were obtained from patients during surgery and were used for RNA extraction. Semi‑quantitative RT‑PCR. Total RNA was extracted from the normal colonic mucosa, fresh surgical tumor and non‑tumor tissue samples using the Eastep Super Total RNA Extraction kit (LS1000, Promega Corp., Shanghai, China), according to the manufacturer's protocol. RNA quantity and quality were assessed using a Synergy HTX Multi‑Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA) and the absorbance ratio of 260/280 nm should have been in the range between 1.8 to 2.0. The integrity of RNA was confirmed using a 2.0% agarose gel electrophoresis and visualized with a Tanon‑2500R Gel Imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China). Electrophoresis of RNA on the agarose gel demonstrated the appearance of a 5S band, and clear 28S and 18S bands that suggested the integrity of RNA. The integrity of RNA was confirmed using agarose gel electrophoresis. First strand cDNA was obtained using the SuperScript II Reverse Transcriptase
EXPERIMENTAL AND THERAPEUTIC MEDICINE 12: 589-596, 2016
591
Table I. Demographic and clinical characteristics of subjects. Characteristic
Healthy controls (n=19)
Non‑tumor controls (n=54)
Early CRC (n=23)
Advanced CRC (n=31)
Mean age (range), years 55 (43‑69) 58 (44‑71) 57 (44‑71) 60 (48‑70) Gender (male/female) 11/8 32/22 13/10 19/12 Tumor location (colon/rectum) N/A 21/33 8/15 13/18 TNM stage (I/II or III/IV) N/A N/A 7/16 (I/II) 21/10 (III/IV) Differentiation (good/moderate/poor) N/A N/A 7/9/7 5/11/15 Serum CEA, ng/ml 0.36 (0.15‑4.58) 7.24 (0.17‑38.34)a 4.37 (0.17‑26.74) 21.16 (2.7‑38.34)a Data are presented as the median and range unless otherwise specified. aP
